The molecular mechanisms by which angiotensin II (ANG II) directly stimulates the activity of the sodium pump (Na,K-ATPase) are unknown any type of cell. Thus, there is a serious gap in our knowledge of how angiotensin-converting enzyme (ACE) inhibitors and ANG II receptor blockers affect cardiovascular function via their effects on sodium transport. For instance, in the proximal tubule, where stimulation of the pump by ANGII is fundamental to the transcellular transport of sodium and water, a 15 min exposure to ANG II has been previously shown to modestly (approximately 20%) stimulate sodium pump activity via activation of the AT1 receptor. Here we show a rapid (< 1 min) robust (5 to 10 fold) and transient direct stimulation at physiological and rate-limiting concentrations of intracellular sodium. Using an innovative ouabain-affinity column and a trypsin digest of the purified a-subunit of the pump, we have shown for the first time that ANG II alters phosphorylation at multiple sites, increasing phosphorylation at two sites, decreasing it at two, with no change at three others. These data support a molecular model in which direct stimulation is mediated by phosphorylation and suggest that the a-subunit contains at least one uncharacterized site of phosphorylation. To test the role of phosphorylation in regulating pump activity we have co-expressed the rat a-subunit in opossum kidney cells, a proximal tubule cell line, along with the AT1A receptor. In these cells ANG II stimulates the activity of the rat sodium pump and alters its phosphorylation at multiple sites in the same pattern seen from the proximal tubule. Using the ouabain-affinity column and site-directed mutagenesis of phosphorylation sites, we will test the hypothesis that ANG II directly and rapidly (<few min) stimulates rat Na,K-ATPase activity through changes in the phosphorylation/dephosphorylation of the a-subunit at both previously known and one or more novel sites as a result of activating the AT1 receptor. Increased activity is due either to alterations in the intrinsic kinetic properties of the pump and/or to its rapid recruitment to the plasma membrane. The specific aims are to: (1) determine the regulatory sites of phosphorylation on the sodium pump through which ANG II controls pump activity and to identify new phosphorylation sites; (2) determine to what extent ANG II stimulates pump activity by altering its kinetic properties compared to its rapid recruitment to the plasma membrane. These results have important implications for understanding normal kidney function, as well as the development of hypertension and heart failure.